Methods and compositions that modulate apoptosis are based on blocking or stimulating components of cell survival or death pathways from NF-κB/IκB through gene activation, to Gadd45β interacting with components of the JNK pathway such as MKK7. Gadd45β-independent JNK modulation exists in certain cell types to regulate apoptosis or cell survival. The JNK pathway is a focus for control of a cell's progress towards survival or death.
Apoptosis or programmed cell death is a physiologic process that plays a central role in normal development and tissue homeostasis. Many factors interact in complex pathways to lead to cell death or cell survival.
A. NF-κB
1. NF-κB in Immune and Inflammatory Responses
NF-κB transcription factors are coordinating regulators of innate and adaptive immune responses. A characteristic of NF-κB is its rapid translocation from cytoplasm to nucleus in response to a large array of extra-cellular signals, among which is tumor necrosis factor (TNFα). NF-κB dimers generally lie dormant in the cytoplasm of unstimulated cells, retained there by inhibitory proteins known as IκBs, and can be activated rapidly by signals that induce the sequential phosphorylation and proteolytic degradation of IκBs. Removal of the inhibitor allows NF-κB to migrate into the cell nucleus and rapidly induce coordinate sets of defense-related genes, such as those encoding numerous cytokines, growth factors, chemokines, adhesion molecules and immune receptors. In evolutionary terms, the association between cellular defense genes and NF-κB dates as far back as half a billion years ago, because it is found in both vertebrates and invertebrates. While in the latter organisms, NF-κB factors are mainly activated by Toll receptors to induce innate defense mechanisms. In vertebrates, these factors are also widely utilized by B and T lymphocytes to mount cellular and tumoral responses to antigens.
Evidence exists for roles of NF-κB in immune and inflammatory responses. This transcription factor also plays a role in widespread human diseases, including autoimmune and chronic inflammatory conditions such as asthma, rheumatoid arthritis, and inflammatory bowel disease. Indeed, the anti-inflammatory and immunosuppressive agents that are most widely used to treat these conditions such as glucocorticoids, aspirin, and gold salts, work primarily by suppressing NF-κB.
TNFα is arguably the most potent pro-inflammatory cytokine and one of the strongest activators of NF-κB. In turn, NF-κB is a potent inducer of TNFα, and this mutual regulation between the cytokine and the transcription factor is the basis for the establishment of a positive feedback loop, which plays a central role in the pathogenesis of septic shock and chronic inflammatory conditions such as rheumatoid arthritis (RA) and inflammatory bowel disease (IBD). Indeed, the standard therapeutic approach in the treatment of these latter disorders consists of the administration of high doses of NF-κB blockers such as aspirin and glucocorticoids, and the inhibition of TNFα by the use of neutralizing antibodies represents an effective tool in the treatment of these conditions. However, chronic treatment with NF-κB inhibitors has considerable side effects, including immunosuppressive effects, and due to the onset of the host immune response, patients rapidly become refractory to the beneficial effects of anti-TNFα neutralizing antibodies.
2. NF-κB and the Control of Apoptosis
In addition to coordinating immune and inflammatory responses, the NF-κB/Rel group of transcription factors controls apoptosis. Apoptosis, that is, programmed cell death (PCD), is a physiologic process that plays a central role in normal development and tissue homeostasis. The hallmark of apoptosis is the active participation of the cell in its own destruction through the execution of an intrinsic suicide program. The key event in this process is the activation by proteolytic cleavage of caspases, a family of evolutionarily conserved proteases. One pathway of caspase activation, or “intrinsic” pathway, is triggered by Bcl-2 family members such as Bax and Bak in response to developmental or environmental cues such as genotoxic agents. The other pathway is initiated by the triggering of “death receptors” (DRs) such as TNF-receptor 1 (TNF-R1), Fas (CD95), and TRAIL-R1 and R2, and depends on the ligand-induced recruitment of adaptor molecules such as TRADD and FADD to these receptors, resulting in caspase activation.
The deregulation of the delicate mechanisms that control cell death can cause serious diseases in humans, including autoimmune disorders and cancer. Indeed, disturbances of apoptosis are just as important to the pathogenesis of cancer as abnormalities in the regulation of the cell cycle. The inactivation of the physiologic apoptotic mechanism also allows tumor cells to escape anti-cancer treatment. This is because chemotherapeutic agents, as well as radiation, ultimately use the apoptotic pathways to kill cancer cells.
Evidence including analyses of various knockout models—suggests that activation of NF-κB is required to antagonize killing cells by numerous apoptotic triggers, including TNFα and TRAIL. Indeed, most cells are completely refractory to TNFα cytotoxicity, unless NF-κB activation or protein synthesis is blocked. Remarkably, the potent pro-survival effects of NF-κB serve a wide range of physiologic processes, including B lymphopoiesis, B- and T-cell co stimulation, bone morphogenesis, and mitogenic responses. The anti-apoptotic function of NF-κB is also crucial to ontogenesis and chemo- and radio-resistance in cancer, as well as to several other pathological conditions.
There is evidence to suggest that JNK is involved in the apoptotic response to TRAIL. First, the apoptotic mechanisms triggered by TRAIL-Rs are similar to those activated by TNF-R1. Second, as with TNF-R1, ligand engagement of TRAIL-Rs leads to potent activation of both JNK and NF-κB. Thirdly, killing by TRAIL is blocked by this activation of NF-κB. Nevertheless, the role of JNK in apoptosis by TRAIL has not been yet demonstrated.
The triggering of TRAIL-Rs has received wide attention as a powerful tool for the treatment of certain cancers, and there are clinical trials involving the administration of TRAIL. This is largely because, unlike normal cells, tumor cells are highly susceptible to TRAIL-induced killing. The selectivity of the cytotoxic effects of TRAIL for tumor cells is due, at least in part, to the presence on normal cells of so-called “decoy receptors”, inactive receptors that effectively associate with TRAIL, thereby preventing it from binding to the signal-transuding DRs, TRAIL-R1 and R2. Decoy receptors are instead expressed at low levels on most cancer cells. Moreover, unlike with FasL and TNFα, systemic administration of TRAIL induces only minor side effects, and overall, is well-tolerated by patients.
Cytoprotection by NF-κB involves activation of pro-survival genes. However, despite investigation, the bases for the NF-κB protective function during oncogenic transformation, cancer chemotherapy, and TNFα stimulation remain poorly understood. With regard to TNF-Rs, protection by NF-κB has been linked to the induction of Bcl-2 family members, BCl-XL and A1/Bfl-1, XIAP, and the simultaneous upregulation of TRAF1/2 and c-IAP1/2. However, TRAF2, c-IAP1, Bcl-XL, and XIAP are not significantly induced by TNFα in various cell types and are found at near-normal levels in several NF-κB deficient cells. Moreover, Bcl-2 family members, XIAP, or the combination of TRAFs and c-IAPs can only partly inhibit PCD in NF-κB null cells. In addition, expression of TRAF1 and A1/Bfl-1 is restricted to certain tissues, and many cell types express TRAF1 in the absence of TRAF2, a factor needed to recruit TRAF1 to TNF-R1. Other putative NF-κB targets, including A20 and IEX-1L, are unable to protect NF-κB deficient cells or were questioned to have anti-apoptotic activity. Hence, these genes cannot fully explain the protective activity of NF-κB.
3. NF-κB in Oncogenesis and Cancer Therapy Resistance
NF-κB plays a role in oncogenesis. Genes encoding members of the NF-κB group, such as p52/p100, Rel, and RelA and the IκB-like protein Bcl-3, are frequently rearranged or amplified in human lymphomas and leukemias. Inactivating mutations of IκBα are found in Hodgkin's lymphoma (HL). NF-κB is also linked to cancer independently of mutations or chromosomal translocation events. Indeed, NF-κB is activated by most viral and cellular oncogene products, including HTLV-I Tax, EBV EBNA2 and LMP-1, SV40 large-T, adenovirus E1A, Bcr-Abl, Her-2/Neu, and oncogenic variants of Ras. Although NF-κB participates in several aspects of oncogenesis, including cancer cell proliferation, the suppression of differentiation, and tumor invasiveness, direct evidence from both in vivo and in vitro models suggests that its control of apoptosis is important to cancer development. In the early stages of cancer, NF-κB suppresses apoptosis associated with transformation by oncogenes. For instance, upon expression of Bcr-Abl or oncogenic variants of Ras—one of the most frequently mutated oncogenes in human tumors—inhibition of NF-κB leads to an apoptotic response rather than to cellular transformation. Tumorigenesis driven by EBV is also inhibited by IκBαM—a super-active form of the NF-κB inhibitor, IκBα. In addition, NF-κB is essential for maintaining survival of a growing list of late stage tumors, including HL, diffuse large B cell lymphoma (DLBCL), multiple myeloma, and a highly invasive, estrogen receptor (ER) in breast cancer. Both primary tissues and cell line models of these malignancies exhibit constitutively high NF-κB activity. Inhibition of this aberrant activity by IκBαM or various other means induces death of these cancerous cells. In ER breast tumors, NF-κB activity is often sustained by PI-3K and Akt1 kinases, activated by over-expression of Her-2/Neu receptors. Constitutive activation of this Her-2/Neu/PI-3K/Akt1/NF-κB pathway has been associated with the hormone-independent growth and survival of these tumors, as well as with their well-known resistance to anti-cancer treatment and their poor prognosis. Due to activation of this pathway cancer cells also become resistant to TNF-R and Fas triggering, which helps them to evade immune surveillance.
Indeed, even in those cancers that do not contain constitutively active NF-κB, activation of the transcription factors by ionizing radiation or chemotherapeutic drugs (e.g. daunorubicin and etoposide) can blunt the ability of cancer therapy to kill tumor cells. In fact, certain tumors can be eliminated in mice with CPT-11 systemic treatment and adenoviral delivery of IκBαM.
B. JNK
1. Roles of JNK in Apoptosis
The c-Jun-N-terminal kinases (JNK1/2/3) are the downstream components of one of the three major groups of mitogen-activated protein kinase (MAPK) cascades found in mammalian cells, with the other two consisting of the extracellular signal-regulated kinases (ERK1/2) and the p38 protein kinases (p38α/β/γ/δ). Each group of kinases is part of a three-module cascade that include a MAPK (JNKs, ERKs, and p38s), which is activated by phosphorylation by a MAPK kinase (MAPKK), which in turn is activated by phosphorylation by a MAPKK kinase (MAPKKK). Whereas activation of ERK has been primarily associated with cell growth and survival, by and large, activation of JNK and p38 have been linked to the induction of apoptosis. Using many cell types, it was shown that persistent activation of JNK induces cell death, and that the blockade of JNK activation by dominant-negative (DN) inhibitors prevents killing by an array of apoptotic stimuli. The role of JNK in apoptosis is also documented by the analyses of mice with targeted disruptions of jnk genes. Mouse embryonic fibroblasts (MEFs) lacking both JNK1 and JNK2 are completely resistant to apoptosis by various stress stimuli, including genotoxic agents, UV radiation, and anisomycin, and jnk3−/− neurons exhibit a severe defect in the apoptotic response to excitotoxins. Moreover, JNK2 was shown to be required for anti-CD3-induced apoptosis in immature thymocytes.
However, while the role of JNK in stress-induced apoptosis is well established, its role in killing by DRs such as TNF-R1, Fas, and TRAIL-Rs has remained elusive. Some initial studies have suggested that JNK is not a critical mediator of DR-induced killing. This was largely based on the observation that, during challenge with TNFα, inhibition of JNK activation by DN mutants of MEKK1—an upstream activator of JNK had no effect on cell survival. In support of this view, it was also noted that despite their resistance to stress-induced apoptosis, JNK null fibroblasts remain sensitive to killing by Fas. In contrast, another early study using DN variants of the JNK kinase, MKK4/SEK1, had instead indicated an important role for JNK in pro-apoptotic signaling by TNF-R.
2. Roles of JNK in Cancer
JNK is potently activated by several chemotherapy drugs and oncogene products such as Bcr-Abl, Her-2/Neu, Src, and oncogenic Ras. Hence, cancer cells must adopt mechanisms to suppress JNK-mediated apoptosis induced by these agents. Indeed, non-redundant components of the JNK pathway (e.g. JNKK1/MKK4) have been identified as candidate tumor suppressors, and the well-characterized tumor suppressor BRCA1 is a potent activator of JNK and depends on JNK to induce death. Some of the biologic functions of JNK are mediated by phosphorylation of the c-Jun oncoprotein at S63 and S73, which stimulates c-Jun transcriptional activity. However, the effects of c-Jun on cellular transformation appear to be largely independent of its activation by JNK. Indeed, knock-in studies have shown that the JNK phospho-acceptor sites of c-Jun are dispensable for transformation by oncogenes, in vitro. Likewise, some of the activities of JNK in transformation and apoptosis, as well as in cell proliferation, are not mediated by c-Jun phosphorylation. For instance, while mutations of the JNK phosphorylation sites of c-Jun can recapitulate the effects of JNK3 ablation in neuronal apoptosis which is dependent on transcriptional events—JNK-mediated apoptosis in MEFs does not require new gene induction by c-Jun. Moreover, JNK also activates JunB and JunD, which act as tumor suppressors, both in vitro and in vivo. Inhibition of JNK in Ras-transformed cells is reported to have no effect on anchorage-independent growth or tissue invasiveness. Hence, JNK and c-Jun likely have independent functions in apoptosis and oncogenesis, and JNK is not required for transformation by oncogenes in some circumstances, but may instead contribute to suppress tumorigenesis. Indeed, the inhibition of JNK might represent a mechanism by which NF-κB promotes oncogenesis and cancer chemoresistance.
C. Biologic Functions of Gadd45 Proteins
Gadd45β (also known as Myd118) is one of three members of the gadd45 family of inducible genes, also including gadd45α (gadd45) and gadd45γ (oig37/cr6/grp17). Gadd45 proteins are regulated primarily at the transcriptional level and have been implicated in several biological functions, including G2/M cell cycle checkpoints and DNA repair. These functions were characterized with Gadd45α and were linked to the ability of this factor to bind to PCNA, core histones, Cdc2 kinase, and p21. Despite sequence similarity to Gadd45α, Gadd45β exhibits somewhat distinct biologic activities, as for instance, it does not appear to participate in negative growth control in most cells. Over-expression of Gadd45 proteins has also been linked to apoptosis in some systems. However, it is not clear that this is a physiologic activity, because in many other systems induction of endogenous Gadd45 proteins is associated with cytoprotection, and expression of exogenous polypeptides does not induce death. Finally, Gadd45 proteins have been shown to associate with MEKK4/MTK1 and have been proposed to be initiators of JNK and p38 signaling. Other reports have concluded that expression of these proteins does not induce JNK or p38 in various cell lines, and that the endogenous products make no contribution to the activation of these kinases by stress. The ability of Gadd45 proteins to bind to MEKK4 supports the existence of a link between these proteins and kinases in the MAPK pathways. Studies using T cell systems, have implicated Gadd45γ in the activation of both JNK and p38, and Gadd45β in the regulation of p38 during cytokines responses.
D. Summary
Although many important cellular processes have been investigated, much is unproven, particularly with respect to the cellular pathways responsible for controlling apoptosis. For example, the manner in which NF-κB controls apoptosis is unclear. Elucidation of the critical pathways responsible for modulation of apoptosis is necessary in order Gadd45β in to develop new therapeutics capable of treating a variety of diseases that are associated with aberrant levels of apoptosis.
Inhibitors of NF-κB are used in combination with standard anti-cancer agents to treat cancer patients, such as patients with HL or multiple myeloma. Yet, therapeutic inhibitors (e.g. glucocorticoids) only achieve partial inhibition of NF-κB and exhibit considerable side effects, which limits their use in humans. A better therapeutic approach might be to employ agents that block, rather than NF-κB, its downstream anti-apoptotic effectors in cancer cells. However, despite investigation, these effectors remain unknown.